Introduction/Background The majority of vulvar squamous cell carcinomas (VSCC) develop independently of human papillomavirus (HPV) and are associated with lichen sclerosus (LS). A small subset of patients with LS progress to VSCC (5%), usually via differentiated vulvar intraepithelial neoplasia (dVIN) which is an aggressive lesion with a high cancer risk (50%). However, dVIN is rarely diagnosed prior to VSCC and accurate diagnosis can be challenging. Our aim was to study the potential value of prognostic DNA methylation biomarkers in vulvar lesions involved in the HPV-independent route towards cancer.

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Abstract 2022-RA-412-ESGO Figure 1

DNA methytation levels (log2 transformed ΔΔCt ratios) increase with increasing severity of disease across six HPV-independent vulvar disease categories, including healthy controls, LS, LS adjacent to VSCC, dVIN, dVIN adjacent to VSCC and VSCC. Differences between categories were tested by the Kruskal-Wallis test, followed by post-hoc testing using the Mann-Whitney U test with Bonferroni multiple testing correction; ****p<0.0001Abbreviations: adj, adjacent; dV1N, differentiated vulvar intraepithelial neoplasia; LS, lichen sclerosus; ns, not significant; VSCC, vulvar squamous cell carcinoma

Methodology A series of 220 HPV-independent vulvar samples were collected, including healthy controls, LS, dVIN, LS adjacent to VSCC, dVIN adjacent to VSCC and VSCC. Samples were tested for 12 DNA methylation markers with quantitative multiplex methylation-specific PCR (qMSP), including genes ASCL1, CADM1, FAM19A4, GHSR, LHX8, MAL, miR124–4, PHACTR3, PRDM14, SST, ZIC1 and ZNF582.

Results Across all twelve markers, significantly higher methylation levels were shown with increasing severity of disease (p<0.001, Kruskal-Wallis test) (figure 1). Comparable low methylation levels were found in healthy vulvar controls and LS samples. Interestingly, LS adjacent to VSCC showed significantly higher methylation levels compared to LS of patients without cancer, whereas none of the markers showed a significant difference in methylation levels between dVIN and dVIN adjacent to VSCC. In fact, methylation levels in dVIN, dVIN adjacent to VSCC and VSCC were consistently high across almost all markers.

Conclusion Our findings indicate the potential of DNA methylation biomarkers to detect HPV-independent precursor lesions with a high cancer risk. As a next step, we aim to further explore these markers in vulvar lesions of patients with a known cancer outcome. Timely identification and treatment of vulvar lesions with a high cancer risk can substantially reduce the risk of malignant progression.